3-4.2.2 Analysis of Southern Blot:

Hybridization of the probe to a specific DNA fragment on the membrane indicates the presence of a complementary fragment in the DNA sequence. Southern hybridization performed by digestion of genomic DNA using a restriction enzyme digestion, helps in determining the number of sequences (or gene copies) in the genome. For a probe hybridizing to a single DNA segment that has not been cut by the restriction enzyme, a single band is observed and on the other hand multiple bands will likely be observed when the hybridization occurs between the probe and several highly similar target sequence (Due to sequence duplication). Alterations in the hybridization conditions like enhancing the hybridization temperature or decreasing salt concentration, helps in altering specificity and hybridization of the probe to sequences that are less than 100% similar.

Fig 3-4.2 .2: Southern hybridization analysis

3-4.2 .3 Applications:

Southern blotting has been exploited for various applications which include:

a) Clone identification: One of the most common applications of Southern blotting is identification and cloning of a specific gene of interest. Southern blotting is carried out for identification of one or more restriction fragments that contain the gene of interest in genomic DNA.. After cloning and tentative identification of the desired recombinant by employing colony or plaque hybridization, southern blotting is further is used to confirm the clone identification and possibly to locate a shorter restriction fragment, containing the sequence of interest.

b) Restriction fragment length polymorphism Analysis: Another major application of Southern hybridization is restriction fragment length polymorphism (RFLP) mapping, which is crucial in construction of genome maps.